Defective regulation of epithelial chloride (C1-) secretion underlies the clinical manifestations of a number of important diseases ranging from cystic fibrosis to the secretory diarrhea associated with enterotoxic infection or intestinal inflammation. While considerable effort has been expended to define the role of apical membrane C1- channels in this process, comparatively little is known about basolateral membrane events. The central hypothesis underlying this research is that the basolateral membrane Na+-K+-2C1-cotransporter (NKCC1) is a critical independent regulatory site that controls net C1- secretory capacity. Accumulating evidence suggests that NKCC1 is regulated at three levels: 1) by phosphorylation-dependent and - independent changes in cotransporter function, 2) by altered cell surface expression, and 3) by long-term changes in gene expression. This project addresses fundamental questions at each level of regulation and utilizes cultured human intestinal epithelial cell lines as model systems. The inhibitory influence of intracellular C1- on NKCC1 activity will be defined, and the potential regulatory roles of the F- actin cytoskeleton and tyrosine kinase signaling on NKCC1 will be assessed. Effects of protein kinase C on F-actin and basolateral membrane recycling will be explored as a possible mechanism for altered NKCC1 surface expression in response to phorbol ester. Finally, long- term changes in NKCC1 gene expression and C1- secretion in response to inflammatory, osmotic, and metabolic stress will be defined. The proposed studies should improve understanding of C1- secretion in the gastrointestinal tract, airway, and exocrine organs by elucidating basic mechanisms of regulation of NKCC1. NKCC1 is expressed in most cells and is thought to play a generalized housekeeping role in controlling cell volume in addition to its role in supporting vectorial transport of ions across polarized epithelia. The long-term objective is to establish a rational basis for designing new therapeutic approaches to disorders of electrolyte transport. Moreover, these studies may have important implications for the regulation of this ubiquitous membrane transport protein in mammalian cells during health and disease.